Protozoa are unicellular eukaryotic organisms that can infect and multiply in mammalian hosts. They may utilize more than one type of host, including insect hosts, during their life cycle. Parasitic protozoa account for a significant portion of all infectious diseases worldwide. Although the majority of protozoan infections occur in developing countries, these infections are seen increasingly in industrialized countries among immigrants and immunosuppressed or immunodeficient individuals. Commonly seen parasitic diseases include malaria, trypanosorriasis, and Chagas disease. The treatment of protozoan infections is problematic due to lack of effective chemotherapeutic agents which traverse the blood brain barrier, excessive toxicity of the therapeutic agents and increasingly widespread resistance to the therapeutic agents. Well known and presently used drugs for treating parasitic infections, caused by protozoa include the drugs melarsopral, eflornithine, chloroquine, quinine, mefloquine, amodiaquine, primaquine, pyrimethamine, sulfadoxine, sulfadiazine, trimethoprim, pentavalent antimony, pentamidine, amphotericin-B, rifampin, metronidazole, ketoconazole, benznidazole, nifurtimox, and halofantrinc.
Two common problems in treatments which involve drugs are drug-toxicity, which debilitates patients, and drug-resistance, which requires more drugs and thus amplifies the problem of drug-toxicity, often resulting in death. One way to solve the problem of drug-toxicity is to deliver drugs so they are targeted only to the infected cells or tissues. Many researchers are working to develop antibodies to deliver drugs, and this approach holds promise, but antibodies are not without problems. For example, they often cross-react with normal tissues, and they can damage blood vessels (e.g., vascular leak syndrome) and cause dangerous allergic reactions (e.g. anaphylaxis).
The treatment of specific cells by the delivery of drugs, including drugs that are toxic to such cells, is not new. U.S. Pat. Nos. 4,886,780; 4,895,714; 5,000,935; and 5,108,987 to Faulk and U.S. Pat. No. 4,590,001 to Stjernholm et. al., describe cytotoxic. or radioimaging materials conjugated to proteins, mainly to transferrin, as treatments for cancerous cells or for imaging cancerous cells.
It is known that stressed cells, such as, for example, human cells hosting a parasitic infection, call for an increased delivery of nutrients, such as iron, by presenting an increased number of receptors for nutrient carriers, such as transferrin in the case of iron. The increase in receptors for nutrient carriers in stressed cells is known to be relatively constant and orders of magnitude greater in number than in unstressed cells, which are known to show receptors intermittently and in relatively smaller numbers. The publications listed above, and others, disclose taking advantage of the increased number of receptors, especially for transferrin, presented by cancer containing cells to deliver imaging materials or drugs or both to the stressed cell.
No single study has asked if all stressed cells have up regulated transferrin receptors, or if all normal cells have down regulated transferrin receptors, but data from many quarters suggest that all normal cells have down regulated transferrin receptors. For example, immature erythrocytes (i.e., normoblasts and reticulocytes) have transferrin receptors on their surfaces, but mature erythrocytes do not (Lesley J, Hyman R, Schulte R and Trotter J. Expression of transferrin receptor on murine hematopoietic progenitors. Cell Immunol 1984; 83: 14–25). Circulating monocytes also do not have up regulated transferrin receptors (Testa U, Pelosi E and Peschle C. The transferrin receptor. Crit Rev Oncogen 1993; 4: 241–276), and macrophages, including Kupffer cells, acquire most of their iron by a transferrin-independent method of erythrophagocytosis (Bothwell T A, Charlton R W, Cook J D and Finch C A. Iron Metabolism in Man, Blackwell Scientific, Oxford, 1979). In fact, in vivo studies indicate that virtually no iron enters the reticuloendothelial system from plasma transferrin (for review, see Ponka P and Lok C N. The transferrin receptor: role in health and disease. Int J Biochem Cell Biol 1999; 31: 1111–1137.). Macrophage transferrin receptors are down regulated by cytokines such as gamma interferon (Hamilton T A, Gray P W and Adams D O. Expression of the transferrin receptor on murine peritoneal macrophages is modulated by in vitro treatment with interferon gamma Cell Immunol 1984; 89: 478–488.), presumably as a mechanism of iron-restriction to kill intracellular parasites (Byrd T F and Horowitz M A. Interferon gamma-activated human monocytes downregulate transferrin receptors and inhibit the intracellular multiplication of Legionella. pneumophila by limiting the availability of iron J Clin Invest 1989; 83: 1457–1465.).
In resting lymphocytes, not only are transferrin receptors down regulated, but the gene for transferrin receptor is not measurable (Kronke M, Leonard W, Depper. J M and Greene W C. Sequential expression of genes involved in human T lymphocyte growth and differentiation. J Exp Med 1985; 161: 1593–1598). In contrast, stimulated lymphocytes up-regulate transferrin receptors in late G1 (Galbraith R M and Galbraith G M. Expression of transferrin receptors on mitogen-stimulated human peripheral blood lymphocytes: relation to cellular activation and related metabolic events. Immunology 1983; 133: 703–710). Receptor expression occurs subsequent to expression of the c-myc proto-oncogene and following up-regulation of IL-2 receptor (Neckers L M and Cossman J. Transferrin receptor induction in mitogen-stimulated human T lymphocytes is required for DNA synthesis and cell division and is regulated by interleukin 2. Proc Nat Acad Sci USA 1983; 80: 3494–3498.), and is accompanied by a measurable increase in iron-regulatory protein binding activity (Testa U, Kuhn L, Petrini M, Quaranta M T, Pelosi E and Peschle C. Differential regulation of iron regulatory element-binding protein(s) in cell extracts of activated lymphocytes versus monocytes-macrophages. J Biol Chem 1991; 266: 3925–3930), which stabilizes transferrin receptor mRNA (Seiser C, Texieira S and Kuhn L C. Interleukin-2-dependent transcriptional and post-transcriptional regulation of transferrin receptor-mRNA. J Biol Chem 1993; 268: 13,074–13,080.). This is true for both T and B lymphocytes (Neckers L M, Yenokida G and James S P. The role of the transferrin receptor in human B lymphocyte activation. J Immunol 1984; 133: 2437–2441), and is an IL-2-dependent response (Neckers L M and Trepel J B. Transferrin-receptor expression and the control of cell growth Cancer Invest 1986; 4: 461–470).
Malaria
Approximately 40% of the world's population are at risk for malaria. That is, in excess of 2000 million people in about 100 countries are at risk (Gilles, 1991, World Health Organization, Geneva). Particularly affected are children in developing countries (Greenwood et al., Trans Soc Trop Med Hyg 1987; 81:478). For example, a million children die of malaria every year in sub-Saharan Africa (World Health Organization, 1974, Technical Report Series No. 537). The rise of travel, trade and tourism also has extended malaria into developed countries (Greenberg & Lobel, Ann Intern Med 1990; 113:326). These social and economic problems are compounded by the complexities of vector control and the problematic development of an effective malaria vaccine (Graves & Gelband, Cochrane Database of Systematic Reviews CD000129, 2000). Thus, anti-malarial drugs remain the bulwark of defense against malaria, but this is being eroded by the spreading emergence of drug resistant strains of Plasmodiurn falciparurn, causing safe, widely available and inexpensive drugs like chloroquine to be increasingly less effective (Clyde, Epidemiol Rev 1987; 9:219). Taken together, these observations-indicate a pressing need for new drug strategies in the war on malaria The present invention provides a new strategy for the design of anti-malarial drugs.
The Plasmodium falciparum parasite reproduces rapidly within red blood cells of its host. Red cells are invaded by the merozoite stage of the parasite, which matures into the trophozoite stage and sufficiently replicates its DNA to produce 32 daughter cells within 48 hours. Like all developing cells (Richardson & Ponka, Biochim Biophys Acta 1997; 1331:1), developing plasmodia require iron to promote the function of key enzymes, such as ribonucleotide reductase for DNA synthesis (Chitambar et al., Biochem J 2000; 345:681), and iron-dependent enzymes for pyrimidine synthesis, CO2 fixation and mitochondrial electron transport (Mabeza et al., Acta Haematol 1996; 95:78). The importance of iron in plasmodial development has been demonstrated in both in vitro Cabantchik et al., Acta Haematol 1996; 95:70) and in vivo (Pollack et al., Proc Soc Exp Biol Med 1987; 184:162) models in which growth of parasites is inhibited by iron chelation. The most widely studied iron chelator is deferoximine, which is a siderophore or chelator that tightly (i.e., affinity of 1031/M) binds iron (Peto & Thompson, Br J Haematol 1986; 63:273). Clinical studies of Zambian children with advanced cerebral malaria (e.g., comatose) have revealed that patients treated with a standard program of anti-malarial therapy plus deferoxamine (100 mg/kg/day) recovered more rapidly than patients who received the same program of anti-malarial therapy without deferoxarine (Gordeuk et al., N Engl J Med 1992; 327:1473).
In light of the key role played by iron in the growth and development of plasmodia, much research has focused on how plasmodia obtain iron, and whether the parasites can be killed by drugs that interfere with the metabolic pathways that are used to acquire iron. Conceptually, plasmodia can obtain iron either from within the red blood cells in which they reside, or from the patient's transferrin, which is the normal protein in blood that carries iron (Ponka & Lok, Int J Biochem Cell Biol 1999; 31:1111). There is little doubt that plasmodia are capable of obtaining iron from red blood cells (Hershko & Peto, J Exp Med 1988; 168:375). In order to obtain iron from the patient's transferrin, there must be transferrin receptors on red blood cells, but normal adult red blood cells do not manifest transferrin receptors (Richardson & Ponka, Biochim Biophys Acta 1997; 1331:1). However, malaria infected red blood cells bind transferrin (Pollack & Fleming, Br J Haematol 1984; 58:289), and data have been produced that have identified 102 kD (Haldar et al., Proc Natl Acad Sci USA 1986; 83:8565) and 93 kD (Rodriguez & Jungery, Nature 1986; 324:388) transferrin receptors in the plasma membranes of red blood cells infected with Plasmodium falciparum. Although these observations have been challenged (Pollack & Schnelle, Br J Haematol 1988; 68: 125), subsequent experiments have shown that the receptors are functional, inasmuch as they have been used to deliver an anti-plasmodial toxin to infected red cells, and such delivery was inhibited by antibody to transferrin (Surolia & Misquith, FEBS Letters 1996; 396:57).
Trypanosomiasis
Trypanosomiasis is a parasitic infection caused by trypanosomes, which are protozoans that are passed to human beings by the bite of an infected tsetse fly (Smith et al., Brit Med Bull 1998; 54:341). When introduced into patients, trypanosomes proliferate in blood and lymphatics, which is the first stage of disease; the second stage of disease develops when parasites traverse the blood-brain-barrier and cause neurological damage and lethargy, commonly known as sleeping sickness (Beutivoglio et al., Trends Neurosci 1994; 17:325). If untreated, trypanosomiasis in both humans and animals is a fatal disease (New York Times, May 21, 2000).
There are two clinical forms of infection that are caused by different trypanosome subspecies. First, Trypanosoma brucei gambiense causes a chronic disease that takes several years to reach advanced stage; second, Trypanosoma brucei rhodesiense causes an acute disease that is fatal within weeks; Both diseases are endemic in Africa, and infections with Trypanosoma brucei gambiense currently are epidemic, placing at risk 60 million people inhabiting 36 sub-Saharan countries (Barrett, Lancet 1999; 353:1113). In addition, trypanosomiasis is limited neither to Africa (Dissanaike, Celyon Med J 2000; 45:40) nor to humans (Karnau et al., Prevent Vet Med 2000; 44:231), and the economic impact of these diseases profoundly impact national economies (Bauer et al., Trop. Animal Hlth & Prod 1999; 31:89).
Diagnostic approaches to trypanosomiasis have been designed to identify the stage of disease in patients, for early infections limited to blood and lymphatics can be treated with less toxic drugs than later infections involving the central nervous system (Dumas & Buiteille, Med Trop 1997; 57:65). There are currently two drugs for treatment of central nervous system infections (i.e., sleeping sickness). The least expensive, most available and most toxic is melarsopral, which is an arsenical drug that induces a fatal encepholopathy in 5–7% of recipients (Harrison et al., Am J Trop Med Hyg 1997; 56:632). These problems are compounded by drug resistance, low response rates and relapse rates as high as 10% (Pepin & Milard, Adv Parasitol 1994; 33:1). A less toxic, more expensive and difficult to acquire alternative to melarsopral is eflornithine, which is an ornithine decarboxylase inhibitor that impedes polyamine synthesis (Sjoerdsma & Schechter, Lancet 1999; 354:254), but this molecule presently is being marketed as an expensive anti-cancer drug.
There also currently are two drugs available for treatment of early stage infections. One of these, pentamidine, was developed in 1941, and the other, suramin, was developed in 1920. Pentamidine also is effective in Pneumocystis carinii infections common in AIDS patients, and it is about 4-fold more expensive than suramin, which for the moment is used only in trypanosomiasis. There are other compounds with trypanocidal activity (Enanga et al., Trop Med Int Health 1998; 3:736), but most of these do not cross the blood-brain-barrier and thus are of limited usefulness in infections of the central nervous system.
The targeted delivery of drugs has the advantage of increasing efficacy while using less drug, thereby decreasing toxicity and causing less damage to normal cells, all of which effectively decrease costs and increase the quality of patient care. Targeted delivery also avoids drug-resistance, which is activated by the non-specific entrance of drugs into cells (Marbeuf-Gueye C, Ettori D, Priebe W, Kozlowski H and Gamier-Suillerot A. Correlation between the kinetics of anthracycline uptake and the resistance factor,in cancer cells expressing the multidrug resistance protein or the P-glycoprotein. Biochem Biophy Acta 1999; 1450: 374–384). Because transferrin-drug conjugates enter cells specifically by employing a receptor-specific pathway (Klausner R D, vanReuswoude J, Ashwell G, Kempf C, Schechter A N, Dean A and Bridges K. Receptor-mediated endocytosis of transferrin in K562 cells. J Biol Chem, 1983; 258: 4715–4724.; Berczi A,.Ruthner M, Szuts V, Fritzer M, Schweinzer E and Goldenberg H. Influence of conjugation of doxorubicin to transferrin on the iron uptake by K562 cells via receptor-mediated endocytosis. Euro J Biochem 1993; 213: 427–436.), they are trafficked around drug-resistance mechanisms, such as efflux pumps in resistant cells.
There exists an unfulfilled need for an inexpensive and effective agent for selectively targeting and eliminating cells diseased by protozoan parasitic invasion